Analysis of mixtures containing different compounds is typically guided by the information desired. For example, analysis of mixtures of chemical compounds may be directed towards identification of said compounds. A general method to identify mixtures constituents is to first separate the mixture and then use one or more techniques for further identification of the individual compounds. Methods of separation depend on the state of the mixture, solid, liquid, or gas, and on known or assumed characteristics of the compounds in the mixture. For example, chromatography is a well known technique to separate mixture components in the liquid or gas phases based on their mass, size, electrical charge, mobility, affinity to other molecules or other properties.
Once separated, many techniques are available for identification of unknown compounds by probing certain physical or chemical properties. For example, spectrophotometric techniques could be used to identify compounds by comparing their absorption spectra to known spectra. Equally important analysis of mixtures of compounds is related to their absolute quantification. Alone or in tandem with their identification, obtaining information regarding the amounts of mixture components is also important for many analytical applications. For example, it is desired to quantify all chemical compounds in a mixture following synthesis, to monitor transformation of a compound over time or after exposure to other compounds, to quantify only the major component of the mixture, or to quantify minor components such as impurities.
In certain fields, chemical analysis is performed in a high throughput manner, and techniques for rapid identification and quantification of both major and minor components of a mixture are highly desired. For example, modem discovery of organic pharmaceutical compounds involves synthesis and screening of thousands or sometimes millions of chemical compounds. Since synthesis techniques are never perfect, compounds that demonstrate initial biological activity are then sent to further chemical analysis. Such analysis involves separation of the assumed mixture of compounds, quantification and identification of all of the constituents. This information could then be used to assess properties of the mixture, such as biological activity, physicochemical parameters such as solubility and lipophilicity and other information relevant to the use of the compound.
While identification of the individual compounds in a mixture can deploy many techniques which probe the chemical composition using multiple physical and chemical aspects, techniques for quantification of said compounds are much more limited in number and scope. Most generally, the compounds in a mixture must be separated and identified. Once separated and identified, standards of the individual components containing known amounts of the mixture are prepared. These standards are typically subjected to further analyses correlating certain physical or chemical properties to their a priori known amounts thus constructing a calibration curve. Finally, the unknown amounts of the components in the mixture are calculated from measuring the same properties previously measured from the standards and using the calibration curve in reverse.
Preparing standards, developing individual calibration curves, and quantifying unknown compounds is a highly resource and time consuming process and not suitable for high throughput analyses. A significant simplification of this process could be achieved if calibration curves could be constructed for a large class of chemical compounds based on a common attribute that could be directly measured. For example, equimolar elemental techniques produce signals which are proportional to the amount of an individual element in the sample. Detectors for quantifying the total carbon, nitrogen or other elements are available. Usually standards containing known amounts of such elements are used to construct calibration curves, which could then be used to analyze any compound containing such elements without the need to prepare an individual series of standards for each unknown compound. In a common scenario, the compound is provided in known volume to the detector which then produces a signal that is proportional to the amount of the element in the sample, thus directly producing a quantitative measure as a total mass or, for example, as parts-per-million or milligrams per milliliter when considering the known volume and properties of the solvent phase. Once the compound is identified, its amount or concentration could be calculated using the number of atoms in the molecule and its molecular weight, for example.
Elemental detectors could achieve an intrinsically very large dynamic range, e.g., from part-per-million to percent concentration of the analyte. However, due to the much more limited range of certain internal components of the detector, for example, a photo-multiplier tube, the overall large dynamic range is realized using sensitivity level selectors. When these detectors are connected to an upstream separation system, a potentially powerful quantification system is possible. For example, a liquid solution containing a mixture of unknown compounds could be injected into a high performance liquid chromatography system which separates the mixture based on any desired property. The eluent from the chromatography column could be directly diverted into an elemental detector and each of the unknown constituent compounds could be quantified and converted to any convenient concentration unit.
In practice, such combination still necessitate constant manual intervention by the analyst, since in most typical applications, the interest is in quantifying a major component in the mixture simultaneously with much smaller amounts of minor components, sometime referred to as impurities. The analyst has to inspect the analysis signal, select alternative sensitivity settings, and repeat the analysis to optimize the signal to noise ratio for each and every desired separated compound. Thus while the entire process is still much simplified, requiring no individual calibration curves to be constructed, the analysis is still cumbersome and requires attention and interim signal evaluation by a trained analyst in order to optimally quantify each component in the mixture.
Devising an automated process which combines both separation and elemental quantification systems, together with a signal analysis and sensitivity gain optimization and control program, is highly desirable, especially for unattended high throughput analyses of mixtures.